Yan-Ru Lou

Antiproliferative Action of Vitamin D

During the past few years, it has become apparent that vitamin D may play an important role in malignant transformation. Epidemiological studies suggest that low vitamin D serum concentration increases especially the risk of hormone-related cancers. Experimentally, vitamin D suppresses the proliferation of normal and malignant cells and induces differentiation and apoptosis. In the present review we discuss the mechanisms whereby vitamin D regulates cell proliferation and whether it could be used in prevention and treatment of hyperproliferative disorders like cancers.

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand.

25-Hydroxyvitamin D(3) 1alpha-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D(3) into 1alpha,25-dihydroxyvitamin D(3), a vitamin D receptor ligand. 25-Hydroxyvitamin D(3) has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1alpha-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D(3) has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D(3) internalisation, in Cyp27b1(-/-) cells explains their higher sensitivity to 25-hydroxyvitamin D(3). 25-Hydroxyvitamin D(3) action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D(3). Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D(3) in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D(3) with 1alpha,25-dihydroxyvitamin D(3) in Cyp27b1(-/-) cells further demonstrates the agonistic action of 25-hydroxyvitamin D(3) and suggests that a synergism between 25-hydroxyvitamin D(3) and 1alpha,25-dihydroxyvitamin D(3) might be physiologically important. In conclusion, 25-hydroxyvitamin D(3) is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.

25-Hydroxyvitamin D3 is an active hormone in human primary prostatic stromal cells

According to the present paradigm, 1α,25‐dihydroxyvitamin D3 [1α,25‐(OH)2D3] is a biologically active hormone; whereas 25‐hydroxyvitamin D3 (25OHD3) is regarded as a prohormone activated through the action of 25‐hydroxyvitamin D3 1α‐hydroxylase (1α‐ hydroxylase). Although the role of vitamin D3 in the regulation of growth and differentiation of prostatic epithelial cells has been well studied, its action and metabolism in prostatic stroma are still largely unknown. We investigated the effects of 25OHD3 and 1α,25‐(OH)2D3 on two human stromal primary cultures termed P29SN and P32S. In a cell proliferation assay, 25OHD3 was found at physiological concentrations of 100–250 nM to inhibit the growth of both primary cultures, whereas 1α,25‐(OH)2D3 at a pharmacological concentration of 10 nM exhibited the growth‐inhibitory effects on P29SN cells but not on P32S cells. Quantitative real‐time RT‐PCR analysis revealed that both 25OHD3 and 1α,25‐(OH)2D3 induced 25‐hydroxyvitamin D3 24‐ hydroxylase (24‐hydroxylase) mRNA in a dose‐ and time‐dependent manner. By inhibiting 1α‐ hydroxylase and/or 24‐hydroxylase enzyme activities, the induction of 24‐hydroxylase mRNA by 250 nM 25OHD3 was clearly enhanced, suggesting that 1α‐hydroxylation is not a prerequisite for the hormonal activity of 25OHD3. Altogether our results suggest that 25OHD3 at a high but physiological concentration acts as an active hormone with respect to vitamin D3 responsive gene regulation and suppression of cell proliferation.

Differentiation of liver progenitor cell line to functional organotypic cultures in 3D nanofibrillar cellulose and hyaluronan-gelatin hydrogels.

Physiologically relevant hepatic cell culture models must be based on three-dimensional (3D) culture of human cells. However, liver cells are generally cultured in two-dimensional (2D) format that deviates from the normal in vivo morphology. We generated 3D culture environment for HepaRG liver progenitor cells using wood-derived nanofibrillar cellulose (NFC) and hyaluronan-gelatin (HG) hydrogels. Culture of undifferentiated HepaRG cells in NFC and HG hydrogels induced formation of 3D multicellular spheroids with apicobasal polarity and functional bile canaliculi-like structures, structural hallmarks of the liver tissue. Furthermore, hepatobiliary drug transporters, MRP2 and MDR1, were localized on the canalicular membranes of the spheroids and vectorial transport of fluorescent probes towards the biliary compartment was demonstrated. Cell culture in 3D hydrogel supported the mRNA expression of hepatocyte markers (albumin and CYP3A4), and metabolic activity of CYP3A4 in the HepaRG cell cultures. On the contrary, the 3D hydrogel cultures with pre-differentiated HepaRG cells showed decreasing expression of albumin and CYP3A4 transcripts as well as CYP3A4 activity. It is concluded that NFC and HG hydrogels expedite the hepatic differentiation of HepaRG liver progenitor cells better than the standard 2D culture environment. This was shown as improved cell morphology, expression and localization of hepatic markers, metabolic activity and vectorial transport. The NFC and HG hydrogels are promising materials for hepatic cell culture and tissue engineering.

The Use of Nanofibrillar Cellulose Hydrogel As a Flexible Three-Dimensional Model to Culture Human Pluripotent Stem Cells

Human embryonic stem cells and induced pluripotent stem cells have great potential in research and therapies. The current in vitro culture systems for human pluripotent stem cells (hPSCs) do not mimic the three-dimensional (3D) in vivo stem cell niche that transiently supports stem cell proliferation and is subject to changes which facilitate subsequent differentiation during development. Here, we demonstrate, for the first time, that a novel plant-derived nanofibrillar cellulose (NFC) hydrogel creates a flexible 3D environment for hPSC culture. The pluripotency of hPSCs cultured in the NFC hydrogel was maintained for 26 days as evidenced by the expression of OCT4, NANOG, and SSEA-4, in vitro embryoid body formation and in vivo teratoma formation. The use of a cellulose enzyme, cellulase, enables easy cell propagation in 3D culture as well as a shift between 3D and two-dimensional cultures. More importantly, the removal of the NFC hydrogel facilitates differentiation while retaining 3D cell organization. Thus, the NFC hydrogel represents a flexible, xeno-free 3D culture system that supports pluripotency and will be useful in hPSC-based drug research and regenerative medicine.

Progressive Hearing Loss in Mice with a Mutated Vitamin D Receptor Gene

Background: Both hypo- and hypervitaminosis D can cause sensorineural hearing loss, and aural symptoms due to vitamin D insufficiency are especially common during gravidity. Hormonal forms of vitamin D regulate transcription by binding with the high-affinity vitamin D receptor (VDR). Objective: To assess the effects of impaired vitamin D action in VDR knockout (KO) mice on hearing, cochlear morphology, and cochlear gene expression. Materials and Methods: Eighteen young male and female mice (10 VDR KO and 8 wild type, WT, ≤6 months old), 33 adult male and female mice (16 VDR KO and 17 WT, between 7 and 14 months old), and 11 aged male and female mice (5 VDR KO and 6 WT, ≧15 months old) on 129S1 genetic background were studied. Auditory thresholds were evaluated by auditory brain stem response. Morphological changes were analyzed using plastic embedding and light microscopy. The expression of key genes (known to play a role in the regulation of cochlear function), and caspase 3 activity, were assessed using immunofluorescent confocal microscopy. Results: There was a statistically significant difference between the young and the adult groups, and between the adult and aged groups of WT mice. There was also a statistically significant difference between the adult and aged groups in VDR KO mice, and between the young WT group and the young VDR KO group. Spiral ganglion cell loss was observed in the basal turn of adult VDR KO mice, a phenomenon infrequently found in WT mice. Expression of connexin 26, KCNJ10, and transient receptor potential channel vanilloid subfamily 4/6 was not affected by VDR KO-mediated hearing loss. Caspase 3 activation was detected in the spiral ganglion cell and its satellite cells, stria vascularis, spiral ligament fibrocytes, and the organ of Corti in both genotypes. However, the percentage of positive cells and the staining intensity were lower in the VDR KO (compared to the WT) mice. Conclusion: These data suggest that sensorineural hearing loss progressively developed at an earlier age in VDR KO mice. While the fundamental gene expressions in the cochlea were not influenced by VDR mutation, it resulted in decrease of caspase 3 activation, which may be one of the factors underlying accelerating age-related hearing loss observed in VDR KO mice.

Inhibition of fatty acid synthase expression by 1α,25-dihydroxyvitamin D3 in prostate cancer cells

Abstract 1α,25-Dihydroxyvitamin D 3 (1α,25(OH) 2 D 3 ) and its derivatives are a potential treatment of human prostate cancer. The antiproliferative action of 1α,25(OH) 2 D 3 is mainly exerted through nuclear vitamin D receptor (VDR)-mediated control of target gene transcription. To explore the target genes which are regulated by 1α,25(OH) 2 D 3 in human prostate cancer LNCaP cells, cDNA microarray was performed by using a chip that contains 3000 gene probes. The results showed that 24 genes were regulated by 1α,25(OH) 2 D 3 . Five of them encode proteins which belong to metabolic enzymes and fatty acid biosynthesis. Fatty acid synthase (FAS) was found to be down-regulated by 1α,25(OH) 2 D 3 , and the regulation was confirmed by real-time quantitative RT-PCR analysis. Inhibition of FAS expression by 1α,25(OH) 2 D 3 in LNCaP cells was more than 50% at 6xa0h. Inhibitory effect of 1α,25(OH) 2 D 3 on FAS expression was completely blocked in the presence of protein synthesis inhibitor cycloheximide, indicating that the down-regulation of FAS gene expression by 1α,25(OH) 2 D 3 was indirect in LNCaP cells. An inhibition of FAS activity by cerulenin resulted in a strong inhibition of LNCaP cell proliferation. The inhibition of FAS expression and cell proliferation by 1α,25(OH) 2 D 3 seemed to be androgen-dependent, since antiandrogen, casodex and DCC-treatment of serum blocked the vitamin D action. The findings suggest that FAS is involved in the antiproliferative effect of 1α,25(OH) 2 D 3 in presence of androgens on prostate cancer LNCaP cells.

Regulation of aromatase and 5α-reductase by 25-hydroxyvitamin D3, 1α,25-dihydroxyvitamin D3, dexamethasone and progesterone in prostate cancer cells

Abstract Estrogens and androgens are proposed to play a role in the pathogenesis of prostate cancer. The effective metabolites, estradiol and 5α-dihydrotestosterone are produced from testosterone by aromatase and 5α-reductase, respectively. Metabolites of vitamin D have shown to inhibit the growth of prostate cancer cells. The aim of the present study was to verify whether 25-hydroxyvitamin D3 (25OHD3), 1α,25-dihydroxyvitamin D3 [1α,25-(OH)2D3], dexamethasone, and progesterone regulate the expression of aromatase and 5α-reductase in human prostate cancer cells. LNCaP and PC3 cells were treated with 25OHD3, 1α,25-(OH)2D3, dexamethasone, or progesterone. Aromatase and 5α-reductase mRNA was quantified by real-time RT-PCR and aromatase enzyme activity was measured by the [3H] water assay. Aromatase enzyme activity in LNCaP and PC3 cells was increased by both 10xa0nM dexamethasone, 1–100xa0nM 1α,25-(OH)2D3 and 100xa0nM–10xa0μM progesterone. The induction was enhanced when hormones were used synergistically. Real-time RT-PCR analysis showed no regulation of the expression of aromatase mRNA by any steroids tested in either LNCaP or PC3 cells. The expression of 5α-reductase type I mRNA was not regulated by 1α,25-(OH)2D3 and no expression of 5α-reductase type II was detected in LNCaP.

A critical assessment of in vitro tissue models for ADME and drug delivery.

Cultured cells are widely used in the evaluation of new drugs and drug delivery systems. Cells can be grown at different levels of complexity ranging from simple reductionist models to complex organotypic models. The models are based on primary, secondary or stem cell derived cell cultures. Generation of tissue mimics with cultured cells is a difficult task, because the tissues have well-defined morphology, complex protein expression patterns and multiple inter-linked functions. Development of organotypic cell culture models requires proper biomaterial matrix and cell culture protocols that are able to guide the cells to the correct phenotype. This review illustrates the critical features of the cell culture models and, then, selected models are discussed in more detail (epidermal, corneal epithelial, retinal pigment epithelium, and hepatocyte models). The cell models are critically evaluated paying attention to the level of characterization and reliability of in vivo translation. Properties of the cell models must be characterized in detail using multiple biological assays and broad sets of model drugs. Robust in vivo predictions can be achieved with well-characterized cell models that are used in combination with computational methods that will bridge the gap between in vitro cell experiments and physiological situation in vivo in the body.

Technetium-99m-labeled nanofibrillar cellulose hydrogel for in vivo drug release

Nanoscale celluloses have recently gained an increasing interest in modern medicine. In this study, we investigated the properties of plant derived nanofibrillar cellulose (NFC) as an injectable drug releasing hydrogel in vivo. We demonstrated a reliable and efficient method of technetium-99m-NFC labeling, which enables us to trace the in vivo localization of the hydrogel. The release and distribution of study compounds from the NFC hydrogel after subcutaneous injection in the pelvic region of BALB/c mice were examined with a multimodality imaging device SPECT/CT. The drug release profiles were simulated by 1-compartmental models of Phoenix® WinNonlin®. The NFC hydrogel remained intact at the injection site during the study. The study compounds are more concentrated at the injection site when administered with the NFC hydrogel compared with saline solutions. In addition, the NFC hydrogel reduced the elimination rate of a large compound, technetium-99m-labeled human serum albumin by 2 folds, but did not alter the release rate of a small compound (123)I-β-CIT (a cocaine analogue). In conclusion, the NFC hydrogels is easily prepared and readily injected, and it has potential use as a matrix for controlled release or local delivery of large compounds. The interactions between NFC and specific therapeutic compounds are possible and should be investigated further.